Hematopoietic stem cells (HSCs) reside in specialized niches and receive signals from surrounding stromal cells, but their identities and the effects they have on HSCs are largely unknown. Our work focuses on defining secreted growth factors that are produced by stromal cells and that trigger HSC expansion ex vivo. We identified a novel cell population from Embryonic Day 15 (E15) fetal livers with the surface phenotype of early T cells and that supports ex vivo expansion of HSCs. Major aims of this grant are to purify and characterize several E15.5 FL T cell subpopulations for their ability to expand HSCs in culture, to determine whether similar cells are found in the adult mouse bone marrow and spleen, to determine whether these are part of the HSC niche, and to examine differences and similarities between gene expression in adult and fetal populations of T cells capable of supporting HSCs in order to develop a cellular and molecular model of HSC expansion. Our DNA microarray experiments showed that, among other proteins, IGF - 2 and Angiopoietin- like proteins 2 (Angptl2) and 3 (AngptIS) are specifically expressed in these E15 FL early T cells. We then developed a serum- free HSC culture system containing low levels of SCF, TPO, FGF-1, IGF- 2 and Angptl2 or AngptIS; as measured by competitive repopulation analyses there was a >24 increase in numbers of long-term repopulating HSCs (LT-HSC) after 10 days of culture. Angptl2 and AngptIS are largely unstudied and have not previously been suggested to act on hematopoiesis or stem cells. Here we will test whether IGF-2, TPO, Angptl2, and other growth factors are each required for maximum HSC expansion ex vivo and thus whether each of these likely activates different signaling pathways in HSCs. In parallel we will adopt a high-resolution cell tracking procedure to determine whether Angptl2, IGF-2, Tpo, and later other factors directly stimulates HSC self- renewal or prevents HSC apoptosis or differentiation. The receptors for Angptl2 or AngptIS and signal transduction pathway(s) are unknown; we will use two expression cloning protocols developed and extensively utilized in my laboratory to isolate the Angptl2 and AngptIS cell surface receptor(s), and then to characterize them extensively. Our studies can lead to major improvements in the clinical use of HSCs in bone marrow transplantation for treatment of leukemia and other cancers; many applications, especially transplantation using cord blood, have been hindered by the low numbers of HSCs and by the unavailability of compatible donors. HSCs are also a promising cell target for developing gene therapies for treating a broad variety of inherited immunodeficiency syndromes and inborn errors of metabolism. The ability to expand HSCs ex vivo would greatly enhance these clinical applications, in the case of gene therapy allowing selection of those HSCs that have integrated a transgene in a specific chromosomal location. [unreadable] [unreadable] [unreadable]